2,4-dichiorophenoxyacetic acid (2,4-D) is in the phenoxy acid class of herbicides and has been used in many monocot crops such as corn, wheat, and rice for the selective control of broadleaf weeds without severely damaging the desired crop plants. 2,4-D is a synthetic auxin derivative that acts to deregulate normal cell-hormone homeostasis and impede balanced, controlled growth; however, the exact mode of action of this class of herbicides is still not fully understood, despite more than 60 years of field application. Triclopyr and fluroxypyr are pyridyloxyacetic acid herbicides that also act as a synthetic auxin.
These herbicides have different levels of selectivity on certain plants (e.g., dicots are more sensitive than monocots). Differential metabolism by different plants is one explanation for varying levels of selectivity. In general, plants metabolize 2,4-D slowly, so varying plant response to 2,4-D may be more likely explained by different activity at the target sites (WSSA, 2002; Herbicide Handbook 8th edition; Weed Science Society of America; Lawrence, Kans. pp. 492.) Plant metabolism of 2,4-D typically occurs via a two-phase mechanism, typically hydroxylation followed by conjugation with amino acids or glucose (WSSA, 2002).
Over time, certain microbial populations challenged with 2,4-D have developed an alternate pathway for degrading this xenobiotic that results in the complete mineralization of 2,4-D. Successive applications of the herbicide select for microbes that can utilize the herbicide as a carbon and energy source for growth, giving them a competitive advantage in the soil. For this reason, currently formulated 2,4-D has a relatively short soil half-life and no significant carryover effects on subsequent crops.
One organism that has been extensively studied for its ability to degrade 2,4-D is Ralstonia eutropha (Streber, et al; 1987; Analysis, cloning, and high-level expression of 2,4-dichlorophenixyacetic monooxygenase gene tfdA of Alcaligenes eutrophus JMP134. J. Bacteriol. 169:2950-2955). The gene encoding the enzyme in the initial step of the mineralization pathway is tfdA. See U.S. Pat. No. 6,153,401 and GENBANK Acc. No. M16730. The TfdA gene product catalyzes the conversion of 2,4-D to dichlorophenol (DCP) via an α-ketoglutarate-dependent dioxygenase reaction (Smejkal; et al.; 2001. Substrate specificity of chlorophenoxyalkanoic acid-degrading bacteria is not dependent upon phylogenetically related tfdA gene types. Biol. Fertil. Sols 33:507-513). DCP has little herbicidal activity compared to 2,4-D. TfdA has been used in transgenic plants to impart 2,4-D resistance in dicot plants such as cotton and tobacco which naturally sensitive to 2,4-D (Streber; et al.; 1989. Transgenic tobacco plants expressing a bacterial detoxifying enzyme are resistant to 2,4-D. Bio/Technology 7:811-816), and U.S. Pat. No. 5,608,147).
A large number of tfdA-type genes that encode enzymes capable of degrading 2,4-D have been isolated from soil bacterial and their sequences deposited into the Genbank database. Many homologues of tfdA (>85% amino acid identity) have similar enzymatic properties to tfdA. However, there are a number of homologues that have a significantly lower identity to tfdA (25-50%), yet have the characteristic residues associated with α-ketoglutarate dioxygenase Fe+2 dioxygenases. Thus it is not obvious what the substrate specificities of these divergent dioxygenases are, among other biochemical properties.
One unique example with low homology to tfdA (28% amino acid identity) is rdpA from Sphingobium herbicidovorans (Kohler, H. P. E. 1999. Sphingobium herbicidovorans MH: a versatile phenoxyalkanoic acid herbicide degrader. J. Ind Microbiol and Biotech. 23:336-340, Westendorf A., D. Benndorf, R. Muller, W. Babel. 2002. The two enantiospecific dichlorprop/α-ketoglutarate-dioxygenases from Delftia acidovorans MC1-protein and sequence data of RdpA and SdpA., Microbiol. Res. 157:317-22). This enzyme has been shown to catalyze the first step in (R)-dichlorprop (and other (R)-phenoxypropionic acids) as well as 2,4-D (a phenoxyacetic acid) mineralization (Westendorf, A., R. H. Muller, and W. Babel. 2003. Purification and characterization of the enantiospecific dioxygenases from Delftia acidovorans MC1 initiating the degradation of phenoxypropionates and phenoxyacetate herbicides. Acta Biotechnol. 23: 3-17). Although the organisms that degrade phenoxypropionic acid were described some time ago, little progress had been made in characterizing this pathway (Horvath, M., G. Ditzelmuller, M. Lodl, and F. Streichsbier 1990). Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium. Appl. Microbiol. Biotechnol. 33:213-216). An additional complication to dichlorprop degradation is the stereospecificity (R vs. S) involved in both the uptake (Kohler, 1999) and initial oxidation of dichlorprop (Westendorf et al., 2003). Heterologous expression of rdpA in other microbes, or transformation of this gene into plants, has not heretofore been reported. Literature has focused mainly on close homologues of tfdA that primarily degrade achiral phenoxyacetic acids (e.g., 2,4-D).
A plant codon-optimized aryloxyalkanoate dioxygenase gene, AAD-1, that encodes the enzyme originally isolated from Delftia acidovorans was first describe for use as a herbicide resistance trait in WO 2005/107437, herein incorporated by reference. The trait confers tolerance to 2,4-D and to pyridyloxyacetate herbicides. The first report of transformed maize hearing the AAD-1 gene was in U.S. Provisional Patent Application No. 61/235,248, herein incorporated by reference.
Companies which develop and market recombinant DNA traits for planting seed products formulate, implement and adhere to strict product stewardship plans. These stewardship plans require the use of validated quantitative and qualitative protein detection methods for the recombinant trait to track trait introgression and seed production activities, as well as monitoring grain harvest for the trait. These detection methods must be facile and robust enough to use under GLP and non-GLP conditions. Moreover the methods must be user friendly and trouble-free enough to be easily employed by farmers in the field, grain dealers at the silo, and customs officials at the borders. Therefore, robust, high quality, user friendly protein detection methods and commercial kits are critical and essential to launching a recombinant plant trait product.
While immunoassays are well-known in the art, developing robust and validated ELISA (enzyme-linked immunosorbent assay) methods that are reproducible and sensitive enough to detect a particular transgenic product in an array of plant tissues in both lab and field settings is neither trivial nor routine. Still more challenging is to find complementary antibody pairs that are suited to the development of lateral flow strip ELISA methods for detecting the product of an AAD-1 transgenic event.